Murine respiratory mycoplasmosis, caused by Mycoplasma pulmonis remains a common problem in not only conventional, but also barrier-maintained rodent colonies. We have developed and extensively tested an enzyme- linked immunosorbent assay (ELISA) for the detection of anti-M. pulmonis antibody. This assay was crucial for establishing the extent of M.. pulmonis infection and gave the first indication that Mycoplasma arthritidis is also common in barrier-maintained colonies. However, there are several limitations of the ELISA antibody detection assay, including the inability to diagnose early, low dose infection when animals do not produce detectable amounts of serum antibody and the inability of ELISA to distinguish between infections with M. pulmonis and M. arthritidis. The long range of this research program is to develop improved methods of detection in order to develop effective control measures. During the previous grant period we have shown which proteins of M. pulmonis were 1) species-specific, 2) genus-common, 3) variable and 4) surface exposed. One of the major surface antigens, V- 1, was shown to be produced both in vivo and in vitro, a prominent antigen recognized by naturally infected animals, and although it contains variable epitopes, it also contains conserved epitopes which are found in all strains of M. pulmonis, and not in the other murine mycoplasmas. It was also shown that this antigen most likely has a subunit structure and contains multiple identical epitopes. Using well characterized monoclonal antibodies to this antigen, we have established an antigen capture assay from M. pulmonis. We have identified a structurally similar antigen on M. arthritidis. Furthermore, we have identified those strain common antigens recognized during low levels of infection and which represent the best candidates for use in an antibody detection assay. A complete genomic library of M. pulmonis has been constructed in E. coli and shown to contain several membrane protein antigens. Also, during the present grant period, we have established the feasibility of using PCR for detection of murine mycoplasmas. Using these reagents, the goals of the present grant period are to standardize and optimize a species-specific, antigen capture assay, antibody detection assay, and PCR for detection of both M. pulmonis and M. arthritidis and to rigorously evaluate the sensitivity and specificity of these assays under both experimental and field conditions. The studies outlined almost surely will meet the main goal of providing a means to detect early , low level infection in barrier-maintained colonies. In addition, they will yield information concerning the membrane proteins of the murine mycoplasmas which will ultimately allow correlation of function and structure for these proteins and provide the necessary background for genetic studies. Other benefits will be the identification of proteins that may prove useful for subunit vaccines. Finally, knowledge gained from these studies should prove useful for similar studies with other pathogens including those of man.